Formation of Acrolein-derived 2�-Deoxyadenosine Adduct in anIron-induced Carcinogenesis Model*□S

Received for publication, August 15, 2003, and in revised form, September 18, 2003Published, JBC Papers in Press, September 30, 2003, DOI 10.1074/jbc.M309057200

Yoshichika Kawai‡, Atsunori Furuhata‡, Shinya Toyokuni§, Yasuaki Aratani¶, and Koji Uchida‡�

From the ‡Laboratory of Food and Biodynamics, Graduate School of Bioagricultural Sciences, Nagoya University,Nagoya 464-8601, Japan, the §Department of Pathology and Biology of Diseases, Graduate School of Medicine, KyotoUniversity, Kyoto 606, Japan, and the ¶Kihara Institute for Biological Research, Yokohama City University,Yokohama 244-0813, Japan

Acrolein is a representative carcinogenic aldehydefound ubiquitously in the environment and formed en-dogenously through oxidation reactions, such as lipidperoxidation and myeloperoxidase-catalyzed aminoacid oxidation. It shows facile reactivity toward DNA toform an exocyclic DNA adduct. To verify the formationof acrolein-derived DNA adduct under oxidative stressin vivo, we raised a novel monoclonal antibody (mAb21)against the acrolein-modified DNA and found that theantibody most significantly recognized an acrolein-modified 2�-deoxyadenosine. On the basis of chemicaland spectroscopic evidence, the major antigenic prod-uct of mAb21 was the 1,N6-propano-2�-deoxyadenosineadduct. The exposure of rat liver epithelial RL34 cells toacrolein resulted in a significant accumulation of theacrolein-2�-deoxyadenosine adduct in the nuclei. Forma-tion of this adduct under oxidative stress in vivo wasimmunohistochemically examined in rats exposed toferric nitrilotriacetate, a carcinogenic iron chelate thatspecifically induces oxidative stress in the kidneys ofrodents. It was observed that the acrolein-2�-deoxyade-nosine adduct was formed in the nuclei of the proximaltubular cells, the target cells of this carcinogenesismodel. The same cells were stained with a monoclonalantibody 5F6 that recognizes an acrolein-lysine adduct,by which cytosolic accumulation of acrolein-modifiedproteins appeared. Similar results were also obtainedfrom myeloperoxidase knockout mice exposed to theiron complex, suggesting that the myeloperoxidase-cat-alyzed oxidation system might not be essential for thegeneration of acrolein in this experimental animal car-cinogenesis model. The data obtained in this study sug-gest that the formation of a carcinogenic aldehydethrough lipid peroxidation may be causally involved inthe pathophysiological effects associated with oxidativestress.

Lipid peroxidation leads to the formation of a broad array ofdifferent products with diverse and powerful biological activi-ties. Among them are a variety of different aldehydes (1). Theprimary products of lipid peroxidation, lipid hydroperoxides(2), can undergo carbon-carbon bond cleavage via alkoxyl rad-icals in the presence of transition metals, giving rise to the

formation of short chain, unesterified aldehydes (2, 3) or asecond class of aldehydes still esterified to the parent lipid.These reactive aldehydic intermediates readily form covalentadducts with cellular macromolecules, including DNA, leadingto disruption of important cellular functions and mutations.The important agents that give rise to the modification of DNAmay be represented by �,�-unsaturated aldehydic intermedi-ates, such as 2-alkenals and 4-hydroxy-2-alkenals (4, 5). 2-Al-kenals represent a group of highly reactive aldehydes contain-ing two electrophilic reaction centers. A partially positive car-bon 1 or 3 in such molecules can attack nucleophiles, such asprotein and DNA, which leads to the formation of cyclic adductsor cross-links. Among all of the �,�-unsaturated aldehydes,acrolein is by far the strongest electrophile (4). Acrolein iswidely found in the environment and is formed in cells via lipidperoxidation (6). Its high reactivity indeed makes this aldehydea dangerous substance for the living cell. A number of reportshave appeared describing the damaging effects of acrolein onthe tracheal ciliatory movement (7) and the pulmonary wall (8).It has also been shown that acrolein reduces the colony-formingefficiency of mammalian cells, forms cyclic adducts withnucleosides in vitro, and is a potent mutagen (4). Moreover,acrolein was shown to initiate urinary bladder carcinogenesisin rats (9).

We have studied the role of reactive aldehydes in the patho-physiological effects associated with oxidative stress. Duringthe course of this study, we found that, upon reaction withprotein, acrolein primarily reacted with lysine residues to forma N�-(3-formyl-3,4-dehydropiperidino)lysine (FDP-lysine)1 ad-duct as the major product (6). In addition, using a monoclonalantibody (mAb5F6) against FDP-lysine, we demonstrated thatthe FDP-lysine adduct recognized by the antibody indeed con-stituted the atherosclerotic lesions, in which intense positivitywas primarily associated with macrophage-derived foam cells(10). Based on these and the in vitro observations (6) thatFDP-lysine was detected in the oxidatively modified low den-sity lipoprotein with Cu2� and that a metal-catalyzed oxidationof arachidonate was associated with the formation of acrolein,we proposed that polyunsaturated fatty acids might representthe potential sources of acrolein under oxidative stress. On theother hand, Anderson et al. (11) have also shown that acrolein

* The costs of publication of this article were defrayed in part by thepayment of page charges. This article must therefore be hereby marked“advertisement” in accordance with 18 U.S.C. Section 1734 solely toindicate this fact.

□S The on-line version of this article (available at http://www.jbc.org)contains supplemental figures.

� To whom correspondence should be addressed. Tel.: 81-52-789-4127;Fax: 81-52-789-5741; E-mail: uchidak@agr.nagoya-u.ac.jp.

1 The abbreviations used are: FDP-lysine, N�-(3-formyl-3,4-dehy-dropiperidino)lysine; Fe3�-NTA, ferric nitrilotriacetate; MPO, my-eloperoxidase; BSA, bovine serum albumin; ELISA, enzyme-linkedimmunosorbent assay; DNPH, 2,4-dinitrophenylhydrazine; PUFA,polyunsaturated fatty acid; LC-MS, liquid chromatography-mass spec-trometry; FITC, fluorescein isothiocyanate; GST, glutathione S-trans-ferase; HPLC, high performance liquid chromatography; mAb,monoclonal antibody; PBS, phosphate-buffered saline; TBARS,2-thiobarbituric acid reactive substances.

THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 278, No. 50, Issue of December 12, pp. 50346–50354, 2003© 2003 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A.

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could be formed from a myeloperoxidase (MPO)-catalyzed oxi-dation of threonine in vitro.

An iron chelate, ferric nitrilotriacetate (Fe3�-NTA), is knownto be a strong inducer of renal proximal tubular necrosis (12). Italso induces a high incidence of renal adenocarcinoma in ro-dents after repeated intraperitoneal administration of Fe3�-NTA (12–14). Oxidative stress has been suggested to play acritical role in the Fe3�-NTA-induced acute nephrotoxicity (15),which finally leads to a high incidence of renal adenocarcinomain rodents (16). In the present study, to verify the formation ofacrolein-derived DNA adduct under oxidative stress in vivo, weraised a new type of monoclonal antibody that specificallyrecognizes an acrolein-derived DNA adduct and examined theformation of this adduct in the Fe3�-NTA-induced carcinogen-esis model. Furthermore, utilizing MPO knockout mice, weexamined whether the MPO-catalyzed oxidation system wasinvolved in the production of acrolein in this experimentalanimal carcinogenesis model.

EXPERIMENTAL PROCEDURES

Materials

Calf thymus DNA, 2�-deoxyribonucleosides, acrolein, and bovine se-rum albumin (BSA) were obtained from Sigma-Aldrich. Polyunsatu-rated fatty acids (PUFAs), such as linoleate, arachidonate, linolenate,cis-5,8,11,14,17-eicosapentaenoic acid, cis-4,7,10,13,16,19-docosahexa-enoic acid, 2,4-dinitrophenylhydrazine (DNPH), and biotin-LC-hydra-zide were obtained from Wako Pure Chemicals Industries (Osaka,Japan). The 50-mer oligonucleoside (dA50, dG50, dT50, and dC50) wereobtained from Hokkaido System Science (Hokkaido, Japan). MPO fromhuman sputum was purchased from Elastin Products Co., Inc. (Owens-ville, MO). Horseradish peroxidase-labeled goat anti-mouse IgG wasobtained from ICN Pharmaceuticals, Inc. (Aurora, OH). An anti-8-oxo-2�-deoxyguanosine monoclonal antibody (mAbN45.1) was obtained fromNOF Co. (Tokyo, Japan). A monoclonal antibody against acrolein-lysineadduct was raised as previously described (6, 10).

Cell Culture

RL34 cells were obtained from the Japanese Cancer Research Re-sources Bank. The cell line, which is a nonmalignant epithelial cell, hasbeen histologically and biochemically shown to possess characteristicsof well differentiated liver parenchymal cells. The cells were grown asmonolayer cultures in Dulbecco’s modified Eagle’s medium supple-mented with 5% heat-inactivated fetal bovine serum, penicillin (100units/ml), streptomycin (100 �g/ml), L-glutamine (588 �g/ml), and0.16% NaHCO3 at 37 °C in an atmosphere of 95% air and 5% CO2. Cellspostconfluency on glass coverslips was exposed to acrolein in Dulbecco’smodified Eagle’s medium containing 5% fetal bovine serum.

General Procedures

NMR spectra were recorded using a Bruker AMX400 (400 MHz)instrument. Fluorescence spectra were recorded with a Hitachi F-2000spectrometer. Fast atom bombardment-mass spectrometry was meas-ured with a JEOL JMS-700 (MStation) instrument. Liquid chromatog-raphy-mass spectrometry (LC-MS) was measured with a Jasco Plat-form II-LC instrument.

Detection of Acrolein in Lipid Peroxidation andMyeloperoxidase-catalyzed Oxidation of Threonine

Auto-oxidation of PUFAs was performed by incubating the emulsi-fied PUFAs liposome (20 mM) with Fe2� (10 �M) and ascorbate (1 mM)in 50 mM sodium phosphate buffer (pH 7.4). The oxidation of threoninewas performed by incubating 200 �M L-threonine with 10 nM MPO, 200�M H2O2, and 150 mM NaCl in 1 ml of 50 mM sodium phosphate buffer(pH 7.4). Aldehydic molecules in the reaction mixtures (100 �l) werederivatized with an equal volume of 0.1% 2,4-dinitrophenylhydrazine(DNPH) in 2 N HCl at room temperature for 30 min. The DNPHderivatives were extracted with chloroform, evaporated, and dissolvedin ethanol. Ten microliters of the aliquot was injected and analyzed ona Develosil ODS-MG-5 column (Nomura Chemicals, Aichi, Japan; 4.6 �250 mm) with a stepwise gradient with water/acetic acid (100/0.01, v/v)(solvent A) and acetonitrile (solvent B) (time � 0, 60% B; 0–30 min, 70%B; 30–35 min, 100% B; 35–55 min, 100% B) at a flow rate of 0.8 ml/min.The elution profiles were monitored by absorbance at 370 nm.

Reaction of 2�-Deoxynucleosides with Acrolein

2�-Deoxynucleosides (2 mM) were incubated with acrolein (10 mM) in0.1 M sodium phosphate buffer (pH 7.4) for 24 h.

Identification of an Antigenic Acrolein-2�-Deoxyadenosine Adduct

The reaction mixture (100 �l) of 2�-deoxyadenosine (2 mM) and acro-lein (10 mM) was injected and fractionated (every 1 min) with a reverse-phase HPLC using a Develosil ODS-HG-5 column (4.6 � 250 mm). Thegradient (flow rate, 0.8 ml/min; solvent A � 5% aqueous acetonitrilecontaining 0.05% acetic acid; solvent B � acetonitrile containing 0.05%acetic acid) was as follows: 0–5 min, 100% A; 5–32 min, linear gradientto 60% B; 32–40 min, linear gradient to 100% B; 40–45 min, lineargradient to 100% A. The elution profiles were monitored by absorbanceat 260 nm. The collected fractions were evaporated and dissolved inPBS, and the immunoreactivity with mAb21 was then examined bycompetitive enzyme-linked immunosorbent assay (ELISA). The majorproduct in the immunoreactive fraction was isolated with a reverse-phase HPLC using a Develosil ODS-HG-5 column (20.0 � 250 mm)equilibrated in 1% acetonitrile containing 0.05% acetic acid at a flowrate of 6.0 ml/min. The chemical structure of the product was charac-terized by mass spectrometry, 1H and 13C NMR (in D2O containingdioxane as an internal standard): �H 2.08 (1H, m, H-8a), 2.18 (1H, m,H-8b), 2.42 (1H, m, H-2�a), 2.67 (1H, m, H-2�b), 3.61 (2H, m, H-5�), 3.97(1H, m, H-4�), 4.25 (1H, m, H-7a), 4.42 (1H, m, H-7b), 4.46 (1H, m, H-3�),5.29 (1H, s, H-9), 6.34 (1H, t, J � 6.56 Hz, H-1�), 8.33 (2H, s, H-2 andH-5); �C 26.86 (C-8), 40.24 (C-2�), 44.46 (C-7), 62.53 (C-5�), 71.35 (C-9),72.03 (C-3�), 86.05 (C-1�), 88.77 (C-4�), 120.22 (C-10b), 144.61 (C-2),147.95 (C-5, C-3a, and C-10a); fast atom bombardment-mass spectrom-etry (�): 308 [M�H]�, 192 [M�H-deoxyribose]�.

Reaction of Calf Thymus DNA with Aldehydes

Calf thymus DNA (1 mg/ml) was mixed with aldehydes (10 mM) andincubated in phosphate buffer (pH 7.4) at 37 °C for 24 h. The reactionwas stopped by ethanol precipitation, and the precipitated DNA waswashed with cold 70% ethanol. The amount of the DNA was determinedby measuring the fluorescence intensity (excitation, 415 nm; emission,505 nm) formed by the reaction of the 2�-deoxyribose moiety with3,5-diaminobenzoic acid.

Preparation of a Monoclonal Antibody againstAcrolein-modified DNA

The acrolein-treated calf thymus DNA (0.5 mg/ml) was electrostati-cally complexed with an equal volume of methylated BSA (0.5 mg/ml)(17). The complex was emulsified with an equal volume of completeFreund’s adjuvant. Six-week-old female Balb/c mice were intraperito-neally immunized with this emulsion (100 �l). After 2 weeks, the micewere boosted with the acrolein-treated DNA/methylated BSA emulsi-fied with an equal volume of incomplete Freund’s adjuvant. In the finalboost, 100 �l of acrolein-treated DNA/methylated BSA in PBS wasintravenously injected. Three days after the final boost, the mouse wassacrificed, and the spleen was removed for fusion with P3/U1 myelomacells. The fusion was carried out by polyethylene glycol, and the cellswere cultured in hypoxanthine/aminopterin/thymidine selection me-dium. After a week, culture supernatants of the hybridomas werescreened by ELISA using acrolein-modified DNA and untreated DNA asthe coating agents. The positive hybridomas were cloned by the limitingdilution method. After repeated screening and cloning, four specificclones were obtained. Among them, a clone (named mAb21) was used inthe following experiment because of its specificity and great ability forcell growth.

ELISA

The indirect noncompetitive ELISA procedure has been previouslydescribed (18). Briefly, 100 �l of antigens in PBS were coated in wellsand kept at 4 °C overnight. After washing and blocking with 4% BlockAce (Dainihon Seiyaku, Osaka, Japan), 100 �l of the mAb21 (1:100dilution) in PBS containing 0.05% Tween 20 (TPBS) was then added,and the wells were incubated at 37 °C for 2 h. After washing, 100 �l ofanti-mouse IgG goat antibody peroxidase labeled (1/5000 in TPBS) wasadded, and the wells were incubated at 37 °C for 1 h. After washing, 100�l of o-phenylenediamine solution (0.5 mg/ml containing 0.03% H2O2 incitrate-phosphate buffer) was added. The color developing reaction wasstopped by the addition of 50 �l of 2 N H2SO4. The binding of theantibody to the antigen was evaluated by measuring the absorbance at492 nm.

Competitive indirect ELISA was performed for estimating the cross-reactivity of the low molecular weight compounds with the antibody.

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The acrolein-modified calf thymus DNA (0.05 �g/well) was used as thecoating antigen. For the competitive reaction, 50 �l of competitors inPBS was mixed with an equal volume of the antibody (1:100 dilution) inPBS containing 4% BSA. The competitor solution was kept at 4 °Covernight, and 90 �l of the mixture was used as the primary antibody.The cross-reactivity of the antibody to the competitors was expressed asB/B0, where B is the amount of mAb21 bound to the coating antigen inthe presence of the competitor, and B0 is the amount in the absence ofa competitor.

Immunocytochemical Detection of Acrolein Adducts

Acrolein-2�-Deoxyadenosine Adduct—The cells were fixed overnightin PBS containing 2% paraformaldehyde and 0.2% picric acid at 4 °C.The membranes were permeabilized by exposing the fixed cells to PBScontaining 0.3% Triton X-100. To eliminate the binding of the antibodyto the RNA containing acrolein-adenosine adduct, coverslips weretreated with 100 �g/ml ribonuclease A (Qiagen) in TEN buffer (10 mM

Tris-HCl, pH 7.4; 1 mM EDTA, pH 7.6; 400 mM NaCl) for 60 min at37 °C. The coverslips were then washed once in PBS and incubated with10 �g/ml proteinase K (Wako) in 100 mM Tris-HCl, pH 7.5, and 10 mM

EDTA for 10 min at room temperature. To provide maximal exposure ofmAb21 to regions of DNA where acrolein-2�-deoxyadenosine lesionsmay exist, the DNA was denatured at room temperature with 2 M HClfor 5 min and then neutralized with 2.5 volumes of 1 M Tris-base. Toprevent nonspecific antibody binding, the coverslips were washed twotimes in PBS and blocked for 1 h at room temperature with 5% BSA inTPBS. The cells were then incubated in primary antibody (mAb21) at4 °C overnight. The cells were then incubated for 1 h in the presence offluorescein isothiocyanate (FITC)-labeled anti-mouse IgG (Dako JapanCo., Ltd., Kyoto, Japan), rinsed with PBS containing 0.3% Triton X-100,and mounted on glass slides using 50% glycerol in PBS. Images ofcellular immunofluorescence were acquired using a Zeiss LSM5PASCAL confocal laser scanning microscope with a 40� objective(excitation, 488 nm; emission, 518 nm). The DNA was stained withpropidium iodide, while the damage was co-localized using a FITC-labeled secondary antibody.

Acrolein-Lysine Adduct—The cells were fixed overnight in PBS con-taining 2% paraformaldehyde and 0.2% picric acid at 4 °C. The mem-branes were permeabilized by exposing the fixed cells to PBS containing0.3% Triton X-100. The cells were then sequentially incubated in PBSsolutions containing blocking serum (5% normal goat serum) and im-munostained with anti-protein-bound acrolein mAb5F6 (10). The cellswere then incubated for 1 h in the presence of FITC-labeled anti-mouseIgG (Dako Japan Co.), rinsed with PBS containing 0.3% Triton X-100and were mounted on glass slides using 50% glycerol in PBS. Images ofcellular immunofluorescence were acquired using a Zeiss LSM5PASCAL confocal laser scanning microscope with a 40� objective (ex-citation, 488 nm; emission, 518 nm). DNA was stained with propidiumiodide, while the damage was co-localized using a FITC-labeledsecondary antibody.

Animal Experiments

Male Wistar rats (Shizuoka Laboratory Animal Center, Shizuoka)weighing 130–150 g (6 weeks old) and mice lacking MPO (MPO�/�mice), the sixth-generation progeny of a backcross into C57BL/6J mice(B6 mice) originally generated by Aratani et al. (19), were used. TheFe3�-NTA solution was prepared immediately before use by the methoddescribed by Toyokuni et al. (15) with a slight modification. Briefly,ferric nitrate enneahydrate and the nitrilotriacetic acid disodium saltwere each dissolved in deionized water to form 80 and 160 mM solutions,respectively. They were mixed at the volume ratio of 1:2 (molar ratio,1:4), and the pH was adjusted with sodium hydrogen carbonate to 7.4.The animals received a single intraperitoneal injection of Fe3�-NTA (15mg of iron/kg of body weight). They were sacrificed by decapitation at 0,24, and 48 h after the administration. Both kidneys of each animal wereimmediately removed. One of them was fixed in Bouin’s solution, em-bedded in paraffin, cut at 3-�m thickness, and used for immunohisto-chemical analyses by an avidin-biotin complex method with alkalinephosphatase. Briefly, after deparaffinization with xylene and ethanol,normal rabbit serum (Dako; diluted to 1:75) for the inhibition of thenonspecific binding of the secondary antibody, a primary antibody (0.5�g/ml), biotin-labeled rabbit anti-mouse IgG serum (Vector Laborato-ries; diluted to 1:300), and avidin-biotin complex (Vector; diluted to1:100) were sequentially used. The procedures using PBS or the IgGfraction (0.5 �g/ml) of normal mouse serum instead of mAb21 showedno or negligible positive responses.

RESULTS

Formation of Acrolein during Peroxidation of PUFAs—Basedon the in vitro observations that the acrolein-lysine adduct(FDP-lysine) was detected in the oxidatively modified low den-sity lipoprotein with Cu2�, PUFAs were suggested to representthe potential sources of acrolein under oxidative stress (6, 10).To evaluate the effectiveness of lipid peroxidation as the originof acrolein, we first examined the generation of acrolein duringin vitro peroxidation of PUFAs, including linoleate, arachido-nate, linolenate, cis-5,8,11,14,17-eicosapentaenoic acid, andcis-4,7,10,13,16,19-docosahexaenoic acid, by a reversed-phaseHPLC following derivatization with DNPH. As shown in Fig.1A, the HPLC analysis of the authentic acrolein showed thatthe DNPH derivative of acrolein was eluted at 32 min (chro-matogram a). The same peak indicated by the arrow was de-tected in the UV chromatogram obtained from the HPLC anal-ysis of the peroxidized arachidonate with Fe2�/ascorbate(chromatogram b). Formation of acrolein was also confirmed byLC-MS analysis following derivatization with biotin-LC-hydra-zide (Fig. S1). A time course analysis of acrolein generation inthe metal-catalyzed peroxidation of PUFAs revealed that sig-nificant amounts of acrolein were generated during peroxida-

FIG. 1. Formation of acrolein during peroxidation of PUFAs.The auto-oxidation of PUFAs was performed by incubating the emulsi-fied PUFAs liposome (20 mM) with Fe2� (10 �M) and ascorbate (1 mM)in 50 mM sodium phosphate buffer (pH 7.4). Aldehydic molecules in thereaction mixtures (100 �l) were derivatized with an equal volume of0.1% DNPH in 2 N HCl at room temperature for 30 min. The DNPHderivatives were extracted with chloroform, evaporated, and dissolvedin ethanol. Ten microliters of the aliquot was injected and analyzed ona Develosil ODS-MG-5 column (Nomura Chemicals, Aichi, Japan; 4.6 �250 mm) with a stepwise gradient with water/acetic acid (100/0.01, v/v)(solvent A) and acetonitrile (solvent B) (time � 0, 60% B; 0–30 min, 70%B; 30–35 min, 100% B; 35–55 min, 100% B) at a flow rate of 0.8 ml/min.The elution profiles were monitored by absorbance at 370 nm. A, HPLCprofiles of authentic acrolein (chromatogram a) and peroxidized arachi-donate (chromatogram b). B, time-dependent formation of acrolein dur-ing peroxidation of PUFAs. E, linolenate; ●, arachidonate; Œ, linoleate;f, cis-4,7,10,13,16,19-docosahexaenoic acid; ‚, cis-5,8,11,14,17-eicosa-pentaenoic acid.

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tion (Fig. 1B), suggesting that lipid peroxidation could repre-sent the major source of acrolein under oxidative stress.

Preparation of a Monoclonal Antibody against Acrolein-mod-ified DNA—It has been suggested that acrolein modification ofDNA may contribute to so-called spontaneous mutagenesis,thereby playing a role in aging and cancer. Hence, to examinewhether acrolein endogenously generated under oxidativestress is involved in the formation of acrolein-DNA adduct, weattempted to detect the acrolein-modified DNA by an immuno-chemical procedure. To raise a monoclonal antibody specific toacrolein-modified DNA, mice were immunized with the acro-lein-modified calf thymus DNA, and after the fusion, the hy-bridomas were selected by the reactivities of the culture super-natant to the immunogen. We finally obtained one clone (cloneNo. 21), which exhibited the most distinctive recognition of theacrolein-modified DNA over native DNA (Fig. 2A). As shown inFig. 2B, incubation of calf thymus DNA (1 mg/ml) with 10 mM

acrolein resulted in a time-dependent increase in immunoreac-tivity with the monoclonal antibody 21 (mAb21). Despite ex-tensive screening of hybridomas, which produce monoclonalantibodies specific to the acrolein-modified DNA, it is stillconceivable that the antibody recognizes epitopes originatingfrom other reactive aldehydes. Hence, we examined the immu-noreactivity of the antibody to aldehyde-treated DNA by adirect ELISA and found that among the aldehydes tested,acrolein was the only source of immunoreactive structuresgenerated in the DNA (Fig. 2C). It is notable that the antibodyscarcely reacted with the DNA treated with an acrolein ana-logue, crotonaldehyde. In addition, the binding of the acrolein-modified DNA to mAb21 was scarcely inhibited by the reactionmixtures of acrolein/2�-deoxyguanosine, acrolein/2�-deoxythy-midine, and acrolein/2�-deoxycytidine but was most signifi-

cantly inhibited by the reaction mixture of acrolein/2�-deoxya-denosine (Fig. 2D). In addition, to attest the selectivityobserved for free acrolein-nucleoside adducts is identical withthe situation in double-stranded DNA, we examined the immu-noreactivity of the acrolein-modified oligonucleoside 50-mers(dA50, dG50, and dC50) by a direct ELISA and found that theantibody reacted only with the acrolein-modified dA50 (Fig. S2).These data strongly suggest that mAb21 selectively recognizesan acrolein-2�-deoxyadenosine adduct as the major epitope.

Identification of an Antigenic Acrolein-2�-DeoxyadenosineAdduct—To identify an acrolein-2�-deoxyadenosine adduct rec-ognized by mAb21, the immunoreactivity with the reactionproducts of acrolein with 2�-deoxyadenosine was characterized.As shown in Fig. 3A, the ELISA analysis of the HPLC fractionsfor immunoreactivity with mAb21 showed that the antibodyhad immunoreactivity with only one fraction (product a) elutedat 4 min. The structure of the antigenic product a was charac-terized by LC-MS, UV, and 1H and 13C NMR. The LC-MSanalysis of the product showed a pseudomolecular ion peak atm/z 308 (M�H)�, suggesting that it was composed of one mol-ecule of 2�-deoxyadenosine and one molecule of acrolein. Fi-nally, the 1H and 13C NMR data (see “Experimental Proce-dures”) determined the antigenic adduct to be 1,N6-propano-2�-deoxyadenosine (Fig. 3B), which was previously identified asthe major acrolein-2�-deoxyadenosine adduct by Smith et al.(20). The isolated 1,N6-propano-2�-deoxyadenosine inhibitedthe antibody binding to the coated antigen in a dose-dependentmanner (Fig. 3C). The reaction of acrolein with other 2�-de-oxynucleosides such as 2�-deoxycytidine and 2�-deoxy-

FIG. 2. Specificity of mAb21. A, immunoreactivity of mAb21 tonative (E) and acrolein-modified DNA (●). Affinity of mAb21 was de-termined by a direct ELISA using acrolein-treated DNA as the absorbedantigen. A coating antigen was prepared by incubating calf thymusDNA (1 mg/ml) with 10 mM acrolein in phosphate buffer (pH 7.4) at37 °C for 24 h. B, time-dependent formation of immunoreactive mate-rials in DNA upon incubation with acrolein. C, immunoreactivity ofmAb21 to the aldehyde-treated DNA. A coating antigen was preparedby incubating calf thymus DNA (1 mg/ml) with 10 mM aldehyde inphosphate buffer (pH 7.4) at 37 °C for 24 h. CRA, crotonaldehyde; HNE,4-hydroxy-2-nonenal; MDA, malondialdehyde. D, competitive ELISAanalysis with the reaction mixtures of 2�-deoxynucleosides and acrolein.The competitors were prepared by incubating 2 mM 2�-deoxynucleosideswith 10 mM acrolein in 0.1 M sodium phosphate buffer (pH 7.4) for 72 h.E, acrolein/2�-deoxythymidine; ●, acrolein/2�-deoxyadenosine. ‚, acro-lein/2�-deoxycytidine; Œ, acrolein/2�-deoxyguanosine.

FIG. 3. Identification of an antigenic acrolein-2�-deoxyade-nosine adduct. A, competitive ELISA analysis of HPLC fractions forimmunoreactivity with mAb21. The reaction was performed by incu-bating 2 mM 2�-deoxyadenosine with 10 mM acrolein in 0.1 M sodiumphosphate buffer (pH 7.4) for 24 h. Upper panel, HPLC profile of UVabsorbance at 240 nm. Lower panel, competitive ELISA analysis. AU,arbitrary units. B, chemical structure of 1,N6-propano-2�-deoxyade-nosine. C, competitive ELISA analysis with 2�-deoxyadenosine (E) and1,N6-propano-2�-deoxyadenosine (●).

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guanosine also gave similar propano-type adducts as the majorproducts, but they scarcely inhibited the binding of the acro-lein-modified DNA to mAb21 (Fig. S3). These data suggest thatthe acrolein-2�-deoxyadenosine adduct (1,N6-propano-2�-de-oxyadenosine) is an intrinsic epitope of mAb21.

Formation of Acrolein-2�-Deoxyadenosine Adduct in RatLiver Epithelial RL34 Cells Exposed to Acrolein—It is knownthat acrolein is extremely toxic to living cells (4). As shown inFig. 4A, the treatment of rat liver epithelial RL34 cells withacrolein resulted in dose- and time-dependent decreases inMTT reduction levels. Essentially all of the cells were detachedfrom culture dishes within 8 h at a dose of 50 �M acrolein (datanot shown). The acrolein-mediated cell death was accompaniedby marked changes in cellular morphology, most notably theswelling of the cells, which changes were evident in phasecontrast microscopy. It was anticipated that the cytotoxicity ofacrolein was due to its facile reactivity toward cellular macro-molecules, including protein and DNA. To examine the corre-lation between cytotoxicity and the generation of theseacrolein-derived adducts, we tested whether the acrolein-2�-deoxyadenosine adduct was formed in cultured cells in vitro. Asshown in Fig. 4B, when the cells were exposed to acrolein (50�M) for 1 h, immunoreactivity with mAb21 was mainly ob-served in the nuclei. In addition, we also observed that acroleingenerated the epitopes recognized by an antibody (mAb5F6)raised against acrolein-modified protein (Fig. 4C). Thus, theacrolein modification of the protein and DNA seems to beclosely associated with the cytotoxicity of acrolein. Meanwhile,we have also observed that acrolein induces depletion of gluta-thione and activation of mitogen-activated protein kinases,such as c-Jun N-terminal kinases and p38, in RL34 cells.2

These cellular events might be associated with apoptotic celldeath. Indeed, Li et al. (21) have reported that acrolein, atlower doses, significantly induces apoptosis of human alveolarmacrophages. These observations suggest that there may be analternative mechanism of acrolein-induced cell death, inde-pendent of modification of protein and DNA.

Formation of Acrolein-2�-Deoxyadenosine Adduct in the Re-nal Proximal Tubules of Rats Exposed to Fe3�-NTA—The for-mation of acrolein-derived epitopes in vivo was immunohisto-chemically assessed in a rat renal carcinogenesis model withFe3�-NTA. The monoclonal antibodies against the acrolein-lysine adduct (mAb5F6) and 8-oxo-2�-deoxyguanosine(mAbN45.1) were also used for comparison. It has been shownthat iron overload using Fe3�-NTA induces acute renal proxi-mal tubular necrosis, a consequence of oxidative tissue dam-age, that eventually leads to a high incidence of renal adeno-carcinoma in rodents (13, 14). The kidneys were excised at thetime of sacrifice and then fixed with Bouin’s fixative. Thehematoxylin and eosin-stained sections of the paraffin-embedded tissues were analyzed for histological damage. Themorphological changes in the kidneys of rats treated with Fe3�-NTA versus time are very similar to previous reports on ddYmice (15, 22). As shown in Fig. 5A, untreated control animalsshowed weak immunoreactivity in all of the proximal tubules.One hour after the administration of Fe3�-NTA (15 mg ofiron/kg of body weight), nuclear stainings were found in some ofthe renal proximal tubular cells in a patchy distribution. In-tense immunoreactivities were also observed at 3 and 24 h.Notably, the proximal tubular cells at higher magnificationrevealed the localization of the acrolein-DNA adduct in thenuclei of degenerating cells (Fig. 5B), suggesting that the for-mation of acrolein and its DNA adduct may be responsible forthe cellular damage. Preabsorption of the antibodies with the

acrolein-modified DNA completely abolished the immunostain-ings (data not shown), indicating the specific reactivity of theseantibodies with their epitopes.2 Y. Kawai, S. Hachisuka, and K. Uchida, unpublished observation.

FIG. 4. Formation of acrolein-2�-deoxyadenosine adduct in ratliver epithelial RL34 cells exposed to acrolein. A, viability of ratliver epithelial RL34 cells exposed to acrolein. Dose-dependent reduc-tion of cell viability induced by 5 �M (�), 10 �M (f), 25 �M (●), and 50�M (E) of acrolein. The cell viability was measured by the MTT assay.The data are expressed as the percentages of control culture conditions.B, immunocytochemical detection of acrolein-2�-deoxyadenosine adductin RL34 cells exposed to acrolein. The cells were incubated with 50 �M

acrolein for 1 h at 37 °C. Panels a–c, control; panels d–f, acrolein-treatedcells. Propidium iodide (PI) (DNA, red) is shown in panels a and d, FITC(acrolein-2�-deoxyadenosine, green) is shown in panels b and e, and thecorresponding combined (superimposed) images are shown in panels cand f (yellow represents co-localization). C, immunocytochemical detec-tion of acrolein-lysine adduct in RL34 cells exposed to acrolein. The cellswere incubated with 100 �M acrolein for 1 h at 37 °C. Panels a–c,control; panels d–f, acrolein-treated cells. Propidium iodide (DNA, red)is shown in panels a and d, FITC (acrolein-lysine, green) is shown inpanels b and e, and the corresponding combined (superimposed) imagesare shown in panels c and f (yellow represents co-localization).

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The pattern of distribution of the acrolein-2�-deoxyadenosineadduct was compared with those of the acrolein-lysine adductand 8-oxo-2�-deoxyguanosine. As shown in Fig. 6, the acrolein-lysine immunoreactivities also appeared in some of the renalproximal tubular cells 1 h after the administration of Fe3�-NTA, whereas their patterns of distribution appeared to besignificantly different. In contrast to the nuclear staining withmAb21, the acrolein-lysine adduct immunoreactive withmAb5F6 was mainly detected in the cytoplasm. This pattern ofdistribution in the rat kidney was consistent with that of thedistribution of other lipid peroxidation products and their con-jugates with cytosolic proteins (15, 23, 24). In addition, theseand the observation that the acrolein-derived adducts co-local-ized with 8-oxo-2�-deoxyguanosine, a marker of oxidativestress, strongly suggest that acrolein was generated underoxidative stress.

MPO-catalyzed Oxidation of Threonine as an AlternativeSource of Acrolein—On the other hand, it has been demon-strated that acrolein is also generated from the MPO-catalyzedoxidation of an amino acid (threonine) in vitro (11). In addition,MPO appeared to catalyze lipid peroxidation (25). These andthe observations that neutrophil and macrophage infiltration ispresent in the kidney of Fe3�-NTA-treated rats3 suggest that

MPO might play a potential role in the generation of acrolein inthis iron-induced carcinogenesis model. Consistent with theprevious findings, acrolein was detected in the MPO-catalyzedoxidation of threonine as the major product (Fig. 7A). A maxi-mal generation of acrolein was observed at 6 h followed by agradual decline to 24 h was observed in the MPO-H2O2-Cl�

system. This transient accumulation of acrolein in the MPO-catalyzed threonine oxidation system was attested to be due tothe secondary reaction of the primary product (acrolein) to thesubstrate (threonine) present in the reaction mixture (data notshown). These data supported the previous findings that MPO-catalyzed threonine oxidation could be a potential source ofacrolein in vivo. To investigate whether MPO is involved in thegeneration of acrolein in the Fe3�-NTA-induced carcinogenesismodel, the MPO-deficient mice were exposed to Fe3�-NTA, andthe formation of the acrolein-derived DNA adduct was immu-nohistochemically examined. The Fe3�-NTA potently inducedlipid peroxidation monitored by 2-thiobarbituric acid reactivesubstances (TBARS) formation in the kidneys of MPO-deficientmice (Fig. 8A). The amount of TBARS reached the peak at 1 hafter administration of Fe3�-NTA (5 mg Fe/kg) and graduallydecreased thereafter. As shown in Fig. 8B, the acrolein-2�-3 S. Toyokuni, unpublished observation.

FIG. 5. Formation of acrolein-2�-deoxyadenosine adduct in therenal proximal tubules of rats exposed to Fe3�-NTA. A, time-de-pendent formation of acrolein-2�-deoxyadenosine adduct (�200). B,higher magnification of the proximal tubules of rats exposed to Fe3�-NTA for 3 h (�400).

FIG. 6. Distribution of acrolein-lysine, acrolein-2�-deoxyade-nosine, and 8-oxo-2�-deoxyguanosine in the kidneys of rats ex-posed to Fe3�-NTA. The formation of acrolein-2�-deoxyadenosine(acrolein-dA) (top panel), acrolein-lysine (middle panel), and 8-oxo-2�-deoxyguanosine (8-Oxo-dG) (bottom panel) immunoreactive withmAb21, mAb5F6, and mAbN45.1, respectively, was immunohisto-chemically assessed in a rat renal carcinogenesis model with Fe3�-NTA(serial sections, �200). The immunoreactivities appeared in some of therenal proximal tubular cells 3 h after the administration of 15 mg ofiron/kg of body weight of Fe3�-NTA. The immunoreactivites withmAb5F6 were mainly detected in the cytoplasm, whereas the immuno-reactivities with mAb21 and mAbN45.1 were mainly detected inthe nuclei.

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deoxyadenosine immunoreactivities appeared in the kidneys ofMPO-deficient mice exposed to Fe3�-NTA. These data providedirect evidence that MPO is not essential for the generation ofacrolein, at least, in this experimental model of carcinogenesis.

DISCUSSION

The previous observations that the metal-catalyzed oxida-tion of low density lipoprotein is associated with the formationof the apo B-bound acrolein suggested that substantialamounts of acrolein might be generated during the peroxida-tion of polyunsaturated fatty acids (6). In the present study, wealso observed that the peroxidation of polyunsaturated fattyacids with an iron/ascorbate-mediated free radical-generatingsystem produces multiple products that react with DNPH, asmonitored by reverse-phase HPLC, and one of the productsco-migrates with the DNPH derivative of authentic acrolein(Fig. 1). These data strongly suggest that the peroxidation ofpolyunsaturated fatty acids represents a potential endogenouspathway for the production of acrolein. Although the mecha-nism for the formation of acrolein during lipid peroxidation hasnot yet been experimentally resolved, it is not unlikely thatacrolein represents a major causal factor that contributes tothe development of tissue damage under oxidative stress.

Among all of the �,�-unsaturated aldehydes, acrolein exhib-its the greatest reactivity with nucleophiles, such as proteins(5). Upon reaction with protein, acrolein selectively reacts withthe side chains of the cysteine, histidine, and lysine residues.

Of these, lysine generates the most stable product. The �-sub-stituted propanals and Schiff’s base cross-links had been sug-gested as the predominant adduct; however, the major adductformed upon the reaction of acrolein with protein was identifiedas a novel lysine product, FDP-lysine, which requires the at-tachment of two acrolein molecules to one lysine side chain (6).In addition, we have recently shown that the FDP-lysine ad-duct generated in the acrolein-modified protein represents athiol-reactive electrophile (26). The formation of a similar FDP-type adduct (dimethyl-FDP-lysine) has been reported in thelysine modification with the acrolein analogue, crotonaldehyde(23). In our previous study, we obtained a murine monoclonalantibody, mAb5F6, that clearly distinguished the acrolein-modified BSA and oxidized low density lipoprotein from nativeBSA and low density lipoprotein and showed that it recognizedthe acrolein-lysine adduct (FDP-lysine) as the major epitope(6). Using this antibody, it was clearly demonstrated that ath-erosclerotic lesions contained antigenic materials in the gran-ular cytoplasmic elements of foam cells and the thickeningneointima of arterial walls (6). In addition, the presence ofantigenic materials has also been observed in the nigral neu-rons of patients with Parkinson’s disease (27), in which oxida-tive stress and mitochondrial respiratory failure with a result-ant energy crisis have been implicated as two majorcontributors to nigral neuronal death.

It has been shown that upon reaction with DNA, acroleinprimarily reacts with 2�-deoxyguanosine and forms the 1,N2-

FIG. 7. Formation of acrolein during myeloperoxidase-cata-lyzed oxidation of threonine. The oxidation of threonine was per-formed by incubating 200 �M L-threonine with 10 nM MPO, 200 �M

H2O2, and 150 mM NaCl in 1 ml of 50 mM sodium phosphate buffer (pH7.4). Aldehydic molecules in the reaction mixtures (100 �l) were deri-vatized with an equal volume of 0.1% DNPH in 2 N HCl at roomtemperature for 30 min. The DNPH derivatives were extracted withchloroform, evaporated, and dissolved in ethanol. Ten microliters of thealiquot was injected and analyzed on a Develosil ODS-MG-5 column(Nomura Chemicals, Aichi, Japan; 4.6 � 250 mm) with a stepwisegradient with water/acetic acid (100/0.01, v/v) (solvent A) and acetoni-trile (solvent B) (time � 0, 60% B; 0–30 min, 70% B; 30–35 min, 100%B; 35–55 min, 100% B) at a flow rate of 0.8 ml/min. The elution profileswere monitored by absorbance at 370 nm. A, HPLC profiles of authenticacrolein (lower panel) and oxidized threonine (upper panel). B, time-de-pendent formation of acrolein during the myeloperoxidase-catalyzedoxidation of threonine.

FIG. 8. Formation of acrolein-derived adducts in the renalproximal tubules of MPO-deficient mice exposed to Fe3�-NTA. A,time-dependent formation TBARS in the kidney of MPO-deficient miceexposed to Fe3�-NTA. B, formation of acrolein-2�-deoxyadenosine ad-duct in the kidney of MPO-deficient mice exposed to Fe3�-NTA for 1 h.Formation of acrolein-lysine and acrolein-2�-deoxyadenosine (acrolein-dA) immunoreactive with mAb5F6 and mAb21, respectively, was im-munohistochemically examined (�200).

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propano-2�-deoxyguanosine adduct (28–30). In later studies,the 1,N2-propano-2�-deoxyguanosine adduct was detected inthe DNA extracted from rat and human liver (31–33) and fromlymphocyte DNA in patients undergoing treatment with cyclo-phosphamide, a precursor of acrolein (34, 35). It is striking tonote that the acrolein-derived 2�-deoxyguanosine adducts aremutagenic in vitro and in vivo (36, 37). This may be correlatedto our previous findings that the acrolein adduct (FDP-lysine)was markedly generated in the livers of rats exposed to thecarcinogenic chemicals, such as butylated hydroxyanisole,4-dimethylaminoazobenzene, and 2-acetylaminofluorene (38).In addition, we confirmed the potency of acrolein to promotethe transformation of benzo[a]pyrene-treated C3H/10T1/2 cellsin vitro (38). These observations raised the possibility thatacrolein generation under oxidative stress could be closely as-sociated with carcinogenesis. Furthermore, the causality ofacrolein in the Fe3�-NTA-induced oxidative tissue damage mayalso be suggested by our previous observation that the Fe3�-NTA exposure to rats dramatically induced gene expression ofGST P1 subunit in the proximal tubular cells (39). Berhane etal. (40) have shown that GST P1 is highly active in catalyzingthe reactions with �,�-unsaturated aldehydes, particularlywith acrolein. In addition, these authors have shown that theGST P1 isozyme introduced in the cells by electroporationincrease their resistance to acrolein. Thus, it can be hypothe-sized that the induction of GST P1 in the proximal tubularcells, the target organs of Fe3�-NTA, may represent the cellu-lar response to the formation of acrolein and its DNA adduct.

To verify the formation of acrolein and to investigate thepotential role of acrolein modification of DNA in vivo, an ex-perimental model of iron overload using Fe3�-NTA was uti-lized. This iron chelate was originally used for an experimentalmodel of iron overload (41). Later, repeated injections of Fe3�-NTA were reported to induce acute and subacute renal proxi-mal tubular necrosis and a subsequent high incidence (60–92%) of renal adenocarcinoma in male rats and mice (13, 14). Asingle injection of Fe3�-NTA causes a number of time-depend-ent morphological alterations in the structure and the functionof the renal proximal tubular cells and their mitochondria.During the early stage of the injury, typical cellular changesare the loss of brush border, cytoplasmic vesicles, mitochon-drial disorganization, and dense cytoplasmic deposits in theproximal tubular cells. Most of the damaged epithelia show thetypical appearance of necrotic cells, and more than half of theproximal tubular cells are removed. It has been suggested thatoxidative stress is one of the basic mechanisms of Fe3�-NTA-induced acute renal injury and is closely associated with renalcarcinogenesis (16). To detect the acrolein-derived DNA ad-ducts in this carcinogenesis model, an immunochemical ap-proach has been taken in this study. Immunologic detection isa powerful tool that can be used to evaluate the presence of adesired target and its subcellular localization. A major advan-tage of this technique over biochemical approaches is the eval-uation of small numbers of cells or archival tissues that mayotherwise not be subject to analysis. We successfully raised amurine monoclonal antibody, mAb21, that clearly distin-guished the acrolein-modified DNA from the native DNA. Thisantibody appeared to be highly specific for the acrolein-modi-fied DNA and did not cross-react with other aldehyde-modifiedDNA (Fig. 2). In addition, we identified the 1,N6-propano-2�-deoxyadenosine as the major epitope of mAb21 (Fig. 3). Usingthis antibody, we demonstrated the formation of the acrolein-2�-deoxyadenosine adduct in rat liver epithelial RL34 cellsexposed to acrolein (Fig. 4). Furthermore, the in vivo studydemonstrated that the Fe3�-NTA treatment resulted in a sig-nificant accumulation of the acrolein adduct in the nuclei of the

proximal tubular cells, the target cells of this carcinogenesismodel (Fig. 5). The same cells were also stained with an alter-native mAb5F6 that recognizes an acrolein-lysine adduct(FDP-lysine), by which cytosolic accumulation of acrolein-mod-ified proteins appeared (Fig. 6). As far as we know, this is thefirst report that immunohistochemically proved the in vivoformation of acrolein-derived DNA adducts in the target organof the carcinogenic protocol.

On the other hand, it has been suggested that MPO in thepresence of H2O2 and Cl� is an alternative source of acrolein invivo (11). MPO is an enzyme found mainly in neutrophils andto a lesser degree in monocytes (42). In chemoattractant-acti-vated neutrophils, MPO transforms H2O2 generated during theoxidative burst into highly cytotoxic hypochlorous acid (HOCl)in the presence of chloride ions (43). This MPO-H2O2-Cl� sys-tem appears to function in the killing of microbes by neutro-phils (19, 44). It may also be involved in their cytotoxicityagainst tumor cells (45, 46) and in the tissue damage at sites ofinflammation where neutrophils can release both MPO andH2O2 (47–50). It has been proposed that the pathway for acro-lein synthesis involves the initial chlorination of the �-aminogroup of threonine by HOCl (11). In the absence of the enzy-matic system, HOCl alone could convert threonine into acro-lein, strongly implicating HOCl in the MPO-H2O2-Cl� system(11). We have also examined the generation of acrolein in thethreonine oxidation and observed that, in contrast to the per-oxidation of polyunsaturated fatty acids, the MPO-H2O2-Cl�

system transiently generated acrolein, which was proven to bedue to the secondary reaction of the product (acrolein) with thecatalyst (MPO) and/or the substrate (threonine) present in thereaction mixture (Fig. 7). Thus, although nonenzymatic lipidperoxidation and MPO-catalyzed threonine oxidation showedconsiderably distinct profiles in kinetics, these data supportedthe previous findings that both systems could be potentialorigins of acrolein in vivo. To investigate whether MPO isinvolved in the generation of acrolein in the Fe3�-NTA-inducedcarcinogenesis model, the MPO-deficient mice were exposed toFe3�-NTA, and the formation of acrolein-derived DNA adductswas immunohistochemically examined. Fe3�-NTA inducedlipid peroxidation monitored by TBARS formation in the kid-neys of MPO-deficient mice (Fig. 8A). The amount of TBARSreached the peak at 1 h after the administration of Fe3�-NTAand gradually decreased thereafter. In parallel with theTBARS formation, the acrolein-2�-deoxyadenosine adductswere significantly accumulated in the kidneys of MPO-deficientmice exposed to Fe3�-NTA for 1 h (Fig. 8B). These data providedirect evidence that MPO is not essential for the generation ofacrolein, at least in this experimental model of renalcarcinogenesis.

In summary, to verify the acrolein generation under oxida-tive stress in vivo, we raised a new type of monoclonal antibody(mAb21) against the acrolein-modified DNA and found that theantibody most significantly recognized the 1,N6-propano-2�-deoxyadenosine adduct. The formation of this adduct was at-tested to in rat liver epithelial RL34 cells exposed to acroleinand in rats exposed to Fe3�-NTA, a carcinogenic iron chelatethat specifically induces oxidative stress in the kidney of ro-dents. These data suggest that the formation of a carcinogenicaldehyde during lipid peroxidation may be causally involved inthe pathophysiological effects associated with oxidative stress.

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Retention time (min)

FeFe2+2+//ascorbateascorbate//ArachidonateArachidonate

AcroleinAcrolein

10 20 30 40

26.8926.89

26.8726.87

B

A

C

FeFe2+2+//ascorbateascorbate//ArachidonateArachidonate

AcroleinAcrolein

FeFe2+2+//ascorbateascorbate//LinoleateLinoleate

Fig. S1. Detection of acrolein in peroxided PUFAs using biotin-LC-hydrazide forderivatization. (A) HPLC profiles of authentic acrolein (top) and peroxidizedarachidonate (middle) and peroxized linoleate (bottom). (B) Mass spectrum of the peakeluted at 32 min in the HPLC chromatogram. (C) Selected ion current chromatogramsobtained from LC-MS analysis monitored with m/z 409 for the biotin-LC-hydrazidederivatives of authentic peroxidized arachidonate (upper) and acrolein (lower). Theautoxidation of PUFAs was performed by incubating the emulsified PUFAs liposome(20 mM) with Fe2+ (10 mM) and ascorbate (1 mM) in 50 mM sodium phosphate buffer(pH 7.4). Aldehydic molecules in the reaction mixtures (100 ml) were derivatized with10 mM biotin-LC-hydrazide in 0.1% TFA at room temperature for 30 min. The formedbiotin-LC-hydrazide derivatives were extracted with chloroform, evaporated, anddissolved in ethanol. Ten microliters of the aliquot was injected and analyzed on aDevelosil ODS-MG-5 column (4.6 x 250 mm) with a linear gradient ofwater/trifluoroacetic acid (100/0.01, by vol.) (solvent A) – acetonitrile/trifluoroaceticacid (100/0.01, by vol.) (solvent B) (time = 0 min, 90% A; 40 min, 60% A), at a flowrate of 0.8 ml/min. The elution profiles were monitored by absorbance at 263 nm.

0

0.4

0.8

1.2

1.6

2.0

2.4

1 10 100

O. D

. 490

nm

Antigen (mg/ml)

Fig. S2. Immunoreactivity of mAb21 to the acrolein-treated oligonucleoside 50mers. A coating antigen was prepared by incubating oligonucleoside 50 mers (dA50,dG50, and dC50) (0.5 mg/ml) with 5 mM acrolein in phosphate buffer (pH 7.4) at 37°Cfor 24 h. Symbols: , dA50; , dG50; , dC50; , acrolein-dA50; , acrolein-dG50; ,acrolein-dC50.

Ab

s. 2

67 n

m

0 2010 155

Acrolein/dA

Acrolein/dG

Acrolein/dC

Acrolein/dT

dA

dC

dG

dT

Propano-dA

Propano-dG

Propano-dC

Retention time (min)

Acrolein

0

0.2

0.4

0.6

0.8

1.0

1.2

10-3 10-2 10-1 100 101

B/B

0

Competitor (mM)

A

N

N

OH

N

N

N

N

O

N

N

NN

N

OH

N

dR

dR

R1

NH

R2

R1 = H, R2 = OHR1 = OH, R2 = Hor

dR

O

Propano-dA Propano-dG Propano-dC

B

Fig. S3. Immunoreactivity of mAb21 to the isolated propano-type acrolein-2’-deoxyadenosine, acrolein-2’-deoxycytidine, acrolein-2’-deoxyguanosine adducts.(A) HPLC profile of the reaction mixtures. The reactions were carried out by incubating2 mM 2’-deoxynucleosides with 10 mM acrolein in 0.1 M sodium phosphate buffer (pH7.4) for 72 h. The mixtures were analyzed by a reverse-phase HPLC using a DevelosilODS-HG-5 column (4.6 x 250 mm) with a linear gradient elution from 5% acetonitrilecontaining 0.05% acetic acid to 60% acetonitrile for 30 min, at a flow rate of 0.8 ml/minStructural characterization of the propane-type adducts was done by LC-MS and 1H-and 13C-NMR. (B) Competitive ELISA analysis with the isolated propano-type acroleinadducts. Symbols: , acrolein-2’-deoxyadenosine (propano-dA); , acrolein-2’-deoxycytidine (propano-dC); , acrolein-2’-deoxyguanosine (propano-dG).

UchidaYoshichika Kawai, Atsunori Furuhata, Shinya Toyokuni, Yasuaki Aratani and Koji

Carcinogenesis Model-Deoxyadenosine Adduct in an Iron-induced′Formation of Acrolein-derived 2

doi: 10.1074/jbc.M309057200 originally published online September 30, 20032003, 278:50346-50354.J. Biol. Chem. 

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